The size of DNA replication intermediates was determined in cultured human lymphoblasts (CCRF-CEM) by alkaline sucrose gradient centrifugation. In thymidine nucleotide deprived cells by the antifolate, methotrexate, DNA intermediates were formed in three distinct sizes. After 1 min pulse with trace quantities of (3H)thymidine (9.5 pmoles/107 cells), the most frequent size was composed of 60 nucleotides chain length which then elongated during a 5-min pulse to DNA of about 265 nucleotides. During pulse-chase experiments these molecules appear to be precursors of much larger DNA molecules sedimenting near the bottom of the gradient. We concluded from these experiments that DNA of about 60 nucleotides are precursors of the 265 nucleotides DNA and that both classes of molecules may comprise a steady state precursor pool for the synthesis of high molecular weight chromosomal DNA. New studies from this laboratory have revealed a hitherto unsuspected effect of l-beta-D-arabinofuranosylcytosine (ara-C), a known antitumor agent in man, on DNA replication in CCRF-CEM cells. Using the method of alkaline sucrose gradient centrifugation, it was found that 5 nM ara-C produces an almost immediate block in the initiation of new DNA molecules followed about 30 min later by a reduction in the rate of elongation of pre-existing DNA molecules. These data, therefore, indicate that overall DNA synthesis is inhibited in two ways by ara-C. First, there is a reduction in initiation frequency of new DNA replicating units and second, a reduction in fork movement. Preliminary results indicate that ara-C is incorporated into nascent DNA and this may contribute to the differential inhibitory effects exerted by this drug on DNA replication in CCRF-CEM cells.